Efficient Inhibition of Transcription Elongation in vitroby Oligonucleotide Phosphoramidates Targeted to Proviral HIV DNA

Abstract

Triplex-forming oligophosphoramidates containing thymines and cytosines or 5-methyl cytosines (5′ T 4CT 4C 6T 3′) bind strongly to a 16 base- pair oligopurine·oligopyrimidine sequence of HIV proviral DNA even at neutral pH. These triple-helical complexes formed with oligonucleotide analogues with N3′ → P5′ phosphoramidate linkages are remarkably stable compared to oligonucleotides with natural phosphodiester linkages. In transcription assays the (T,C)-containing phosphoramidate oligomers induce an efficient arrest of both bacteriophage and eukaryotic transcrip- tional machineries under conditions where the isosequential phosphodi esters have no inhibitory effect. In both cases the RNA polymerase (SP6, T7 or Pol II) is physically blocked by the non-covalent triplex and RNA synthesis is stopped at the triplex site. However the eukaryotic transcription machinery is blocked more efficiently (at submicromolar concen tration) than the bacteriophage polymerases. The analysis of the 3′-ends of the truncated transcripts provides evidence for differences in the termination patterns induced by the triplex barrier for the bacteriophage and the eukaryotic systems. This in vitrocomparative study provides the basis for the rational design of strong transcriptional inhibitors. The efficient in vitroinhibition obtained using the phosphoramidate oligomers in the eukary- otic transcription assay makes them good candidates for the development of sequence-specific antigene agents.